Chloroplast genome analysis and genetic transformation of Parachlorella kessleri-I a potential marine alga for biofuel production

Abstract

Background: Parachlorella kessleri-I produces higher biomass and lipid content suitable for commercial production of biofuels. Sequencing complete chloroplast genome will be instrumental in the constructing species specific chloroplast transformation vectors and generating chloroplast transgenic microalga with the desired traits and greater productivity, essential for commercial sustainability of microalgae based biofuel production. Results: Complete chloroplast genome sequence (cpDNA) of P. kessleri-I was annotated. The 109,642 bp chloroplast genome exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC) and a small single copy (SSC) region. The genome encodes 117 unique genes, with 70 predicted protein coding genes, 35 tRNAs, 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species specific chloroplast transformation vector that facilitated chloroplast transformation of P. kessleri-I. To optimize chloroplast transformation, two antibiotic resistance makers aminoglycoside adenine transferase (aadA) conferring resistance to spectinomycin and Sh-ble gene from bacteria that conferred resistance to zeocin were tested. Using a aadA gene, transgenic colonies were retrieved on TAP medium containing 400 mg/l spectinomycin. However, no transgenic colonies were recovered in the zeocin supplemented medium. The spectinomycin resistant algal cell lines were analyzed by PCR. Southern blotting confirmed the stable transgenes integration into the chloroplast genome of P. kessleri-I via homologous recombination. Conclusion: The complete chloroplast genome analysis may provide valuable resources for population and evolutionary studies of Parachlorella species and identifying the related species. The chloroplast genome of P. kessleri-I was assembled as a quadripartite structure of 109,642 bp with defined IR regions. Its complete sequencing has provided essential information like codons, UTRs and flanking sequences to generate the species specific chloroplast transformation vector and obtaining the successful site-specific chloroplast transformation in P. kessleri-I via homologous recombination. The optimized chloroplast transformation in marine alga P. kessleri-I should open a new possibilities like controlling the chain lengths of fatty acids for biofuel application, manipulating the RubisCo enzyme for improving photosynthetic process, introducing carbon concentration mechanism (CCM) for fixing higher CO2 that may be instrumental in producing economically viable biofuel molecules.